The goals of this project are to characterize the capacities of various alcohol dehydrogenases (ADHs) to catalyze the oxidation of aldehydes to the corresponding acids and to conduct sequential oxidations of alcohols to the carboxylic acids. Dr. Oppenheimer has obtained data which indicate that the efficiency of the aldehyde oxidation activity catalyzed by ADH from horse liver has likely been grossly underestimated due to reliance on the A340 assay, interference from amine buffers, and failures to consider the complications that arise from a dismutation reaction. Using less ambiguous assays, such as NMR, the principal investigator will monitor concentrations of products and reactants to construct progress curves that will be analyzed by kinetic simulations to assess the relative efficiencies of alcohol and aldehyde oxidations. He will conduct similar investigations of the ADH from Drosophila and of lactate dehydrogenase (LDH)--two enzymes which differ from the HL (horse liver) ADH mechanistically and stereochemically. As a final test of the generality of the aldehyde dehydrogenase activity of ADHs, the principal investigator proposes to replace the "active site thiol" of benzaldehyde dehydrogenase by site directed mutagenesis in an attempt to convert this enzyme into an ADH. Although the ADHs are seemingly well-characterized enzymes, recognition of the capacity of these enzymes to catalyze oxidation of their product aldehydes can bring about an understanding of unusual properties and unexplained observations that have been reported for these enzymes. The results from these studies will likely have an impact on understanding of questions related to ethanol metabolism, the evolution of dehydrogenases and the mechanism of hydride transfer.